Abstract
The F-domain of rat HNF-4alpha1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4alpha. (i) Fluorescent binding assays indicate that wild-type, full-length HNF-4alpha (amino acids 1-455) has high affinity (Kd=0.06-12 nm) for long chain fatty acyl-CoAs (LCFA-CoA) and low affinity (Kd=58-296 nm) for unesterified long chain fatty acids (LCFAs). LCFA-CoA binding was due to close molecular interaction as shown by fluorescence resonance energy transfer (FRET) from full-length HNF-4alpha tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor), which yielded an intermolecular distance of 33 A, although no FRET to cis-parinaric acid was detected. (ii) Deleting the N-terminal A-D-domains, comprising the AF1 and DNA binding functions, only slightly affected affinities for LCFA-CoAs (Kd=0.9-4 nm) and LCFAs (Kd=93-581 nm). (iii) Further deletion of the F-domain robustly reduced affinities for LCFA-CoA and reversed ligand specificity (i.e. high affinity for LCFAs (Kd=1.5-32 nm) and low affinity for LCFA-CoAs (Kd=54-302 nm)). No FRET from HNF-4alpha-E (amino acids 132-370) tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor) was detected, whereas an intermolecular distance of 28 A was calculated from FRET between HNF-4alpha-E and cis-parinaric acid. (iv) Circular dichroism showed that LCFA-CoA, but not LCFA, altered the secondary structure of HNF-4alpha only when the F-domain was present. (v) cis-Parinaric acid bound to HNF-4alpha with intact F-domain was readily displaceable by S-hexadecyl-CoA, a nonhydrolyzable thioether analogue of LCFA-CoAs. Truncation of the F-domain significantly decreased cis-parinaric acid displacement. Hence, the C-terminal F-domain of HNF-4alpha regulated ligand affinity, ligand specificity, and ligand-induced conformational change of HNF-4alpha. Thus, characteristics of F-domain-truncated mutants may not reflect the properties of full-length HNF-4alpha.
Highlights
Hepatocyte nuclear factor-4␣ (HNF-4␣)1 [1] is a member of the superfamily of nuclear receptors highly expressed in liver, intestine, pancreas, and kidney, where it modulates transcription of multiple genes involved in lipid and glucose homeostasis [2,3,4]
1 The abbreviations used are: HNF-4␣, hepatocyte nuclear factor-4␣; aa, amino acid(s); AR, androgen receptor; CoA, coenzyme A; FRET, fluorescence resonance energy transfer; long chain fatty acids (LCFA), long chain fatty acid; intestine, pancreas, and kidney, where it modulates transcription of multiple genes involved in lipid and glucose homeostasis [2,3,4]
The reported affinities based on this radioligand competition binding assay were low, thereby suggesting that neither LCFA-CoA nor LCFA were physiologically significant ligands for HNF-4␣ [12, 13, 16]
Summary
Chemicals—Nonfluorescent long chain fatty acids (C14:0, myristic; C16:0, palmitic; C16:1, palmitoleic; C20:4, arachidonic) and long chain fatty acyl-CoAs (C14:0-CoA, myristoyl-CoA; C16:0-CoA, palmitoyl-CoA; C16:1-CoA, palmitoleoyl-CoA; C20:4-CoA, arachidonyl-CoA) were purchased from Sigma. Calculation of binding parameters (i.e. dissociation constant, Kd, and n (number of binding sites)) was performed from the titration curves plotted as quenching in protein fluorescence intensity (F0 Ϫ F, where F0 and F represented the HNF-4␣ Tyr/Trp fluorescence intensities in the absence and presence of ligand, respectively, at each titration point) as a function of ligand concentration. Linear plots of 1/(1 Ϫ Fi/Fmax) versus [ligand]/(Fi/Fmax) according to Equation 1 were used for calculation of Kd and n values In this case, Fi and Fmax measured the decrease in protein fluorescence intensity (i.e. quenching) in the presence of ligand at each titration point and at saturation, respectively [19]. Controls were run for buffer alone or for ligand in buffer in the absence of protein, and their spectra were subtracted from the actual protein-containing sample spectra
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